Project Summary The HIV-1 Tat transactivator interacts with P-TEFb (Cyclin T1:CDK9) and the AFF4:AFF1:AF9:ENL:ELL2 complex to stimulate elongation at the HIV-1 promoter. It was recently shown that Tat also recruits the SKIP splicing/elongation factor and MLL1:Menin H3K4 methyltransferase, assembling a multicomponent complex similar to that used by leukemogenic MLL activators. Here, novel components of the Tat and SKIP complexes (Aim 1) are identified, and their function in HIV-1 elongation, mRNA splicing, and virus replication (Aim 2) assessed. Residues in SKIP critical for HIV-1 splicing and transcription will be defined, and the role of 3' splice site recognition factors in pausing and elongation at the HIV-1 promoter (Aim 3) will be examined. In the cell, SKIP is required for expression of the anti-apoptotic p21Cip1 protein, a potent inhibitor of HIV-1 replication. It will be assessed whether Tat blocks the normal anti-apoptotic function of SKIP, thereby downregulating p21Cip1 expression and inducing p53-mediated T cell apoptosis. Tat-induced apoptosis will be characterized in the wild-type and isogenic p53-/- HCT116 cell lines, a stable SKIP expressing cell line that strongly resists p53 apoptosis, and in primary CD4+ T cells (Aim 4). Together, these experiments will better define the Tat transactivation mechanism and its role in p21Cip1 downregulation and T cell apoptosis.